The optical relay system consists of several lenses. It can be constructed symmetrically with the result that the imaging by the optical relay system takes place on a scale of 1:1. However, this is not absolutely necessary; the imaging can also take place with magnification or demagnification.
Such a device is used in particular in the examination of biological samples, in which the illumination of the sample is carried out with a light sheet, the plane of which intersects the optical axis of detection at an angle different from zero. Typically here, the light sheet with the detection direction includes a right angle. With this technique, also referred to as SPIM (Selective Plane Illumination Microscopy), three-dimensional recordings even of thicker samples can be generated within a relatively short period of time. On the basis of optical sections combined with a relative movement in a direction perpendicular to the section plane, an image representation, which is expanded three-dimensionally, of the sample is possible.
The SPIM technique is preferably used in fluorescence microscopy, where it is then also referred to as LSFM (Light Sheet Fluorescence Microscopy). Compared with other established methods such as confocal laser scanning microscopy or two-photon microscopy, the LSFM technique has several advantages: since the detection can take place in the wide field, larger sample areas can be detected. In addition, the exposure of the samples to light is the lowest in this method, which among other things reduces the risk of bleaching of a sample since the sample is only illuminated by a thin light sheet at an angle to the detection direction which is different from zero. Instead of a purely static light sheet, a quasi-static light sheet can also be used. This is generated by rapidly scanning the sample with a light beam. The light-sheet-like illumination is produced when the light beam is subjected to a very rapid movement relative to the sample to be viewed and in the process is repeated in a temporal succession so as to be lined up side by side. Here, the integration time of the camera, on the sensor of which the sample is imaged, is chosen appropriately such that the scanning is completed within the integration time.
The SPIM technique has in the meantime been described on many occasions in the literature, for example in DE 102 57 423 A1 and WO 2004/0535558 A1 based thereon. Methods and arrangements by means of which a particularly thin light sheet can be constructed are described for example in DE 10 2012 013 163.1.
In the conventional SPIM arrangements, the illumination is carried out via a lens system which lies in the plane of the sample that is being illuminated. If the sample is thus for example viewed from above, the illumination has to be carried out from the side. Conventional preparation techniques can therefore not be used. A further fundamental disadvantage lies in the fact that both the illumination objective and the viewing objective have to be arranged spatially close to each other, with the result that a lens with a high numerical aperture which captures light from a wide area can be used for the detection. At the same time, however, a light sheet must also be generated. These mechanical limitations can lead to the numerical aperture and thus the resolution of the imaging system being restricted.
In order to overcome these limitations, SPIM optical systems have been developed in which the same objective is used for the illumination with a light sheet and simultaneously for the detection of fluorescence which comes from the sample. Here, the sample is illuminated with a light sheet via a partial area of the objective which includes an edge area of this objective, with the result that the illumination is thus carried out at an angle which is inclined relative to the optical axis of the objective. An opposite edge area of the objective is then used for the detection, with the result that the detection takes place in the centre likewise at an angle to the optical axis of the objective which is different from zero. As a result of the limited numerical aperture of the objective this angle is as a rule less than 90°, which is usual in the classical SPIM technique.
Such a setup is described for example in US 2011/0261446 A1. The imaging system therein is complemented by a relay system which consists of the mirror-symmetrical coupling together of two imaging subsystems. The two imaging systems are arranged mirror-symmetrically with regard to their optical elements, wherein the mirror plane corresponds to the original image plane of the object-side subsystem in which the illuminated area of the sample in the image thus intersects the image plane at an angle. The magnification of the relay system is chosen such that it corresponds to the ratio of the refractive indices of a first medium, in which the sample is located, to a second medium, in which the intermediate image is located.
If no immersion media are used, the optical components of the two subsystems can be chosen to be identical; however they are arranged in a mirror-inverted manner with the result that the imaging takes place on a 1:1 scale.
If one of the two subsystems is designed as an immersion system, the optical element which is closest to the sample is thus located in an immersion medium, and consequently according to US 2011/0261446 A1 magnifications should be chosen which correspond to the ratio of the refractive indices of the object-side medium and of an image-side medium or immersion medium. Using the optical relay system, which is symmetrical except for the use of immersion media, the object plane is thus imaged into an intermediate image in an intermediate image plane, wherein the intermediate image plane again coincides with the light sheet plane with the result that the object plane is represented undistorted and unmagnified relative to the intermediate image plane.
In order then to obtain a magnified representation of the sample in the object plane, US 2011/0261446 A1 provides an optical imaging system designed as a microscope which comprises an objective, the optical axis of which lies perpendicularly on the intermediate image plane. It is also focused on the intermediate image plane, and the focal planes of the relay system and of the imaging system intersect in the centre of the intermediate image. In this way an undistorted imaging of the sample, that is to say an imaging which is free from aberrations, can take place onto a detector with a magnification which is dependent on the microscope. The underlying principle is also described in WO 2008/078083 A1 according to which, using such a system, an object can be imaged at depth in a certain volume range in an image plane lying perpendicular to the optical axis without comatic aberration and spherical aberration.
Instead of a transmissive relay system composed of lenses, as is used in US 2011/0261446 A1, a partially catadioptric, i.e. reflectively operating system can also be used. Such a system is described for example in the previously unpublished DE 10 2013 105 586.9, by means of which the overall length and number of optical elements can be reduced.
It can be learned from US 2011/0261446 A1 that the angular distribution of the fluorescent beams to be detected in the plane spanned by the light sheet illumination direction and the optical axis of the relay system is symmetrical relative to the propagated optical axis of the optical imaging system which is downstream of the relay system. In the detection pupil there is no overlapping between excitation and detection beams. The numerical aperture of the optical imaging system which is downstream of the relay system also limits the angular spectrum of the sample that can be detected. Here, the numerical aperture of the relay system on the object side is larger than the numerical aperture of the optical imaging system.
A similar arrangement is described in DE 10 2011 000 835 A1 wherein the light sheet is generated by means of a scanning movement; it is thus a quasi-static light sheet. Here too, the angular spectrum of the detection beam path is arranged symmetrically relative to the optical axis of the optical imaging system which is downstream of the relay system, and the range of the angular spectrum here is also limited by the numerical aperture of the optical imaging system.